Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real-time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10-100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra- and inter-assay variations of each assay were < 9.4 and < 12.6%, respectively. Therefore, the real-time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non-fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non-fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non-fibrolytics.